This invention relates to a method for the recovery of refined .alpha.-galactosidase. More particularly, the present invention relates to a method for recovering .alpha.-galactosidase of high purity from an .alpha.-galactosidase-containing culture broth obtained by the culture of an .alpha.-galactosidase-producing microorganism or from an .alpha.-galactosidase-containing liquid obtained by the extraction of .alpha.-galactosidase from the microorganic cells after said culture.
Today it is known that .alpha.-galactosidase is produced by microorganisms such s actinomycetes, molds, etc. It is an enzyme which has the ability to hydrolyze raffinose into sucrose and glactose. In the beet sugar production industry, this enzyme is employed to hydrolyze the raffinose present in beet sugar solution such as beet juice and beet molasses so as to improve the yield of sucrose. Furthermore, .alpha.-galactosidase refined to high purity is used as a structure-determining agent in the chemical and biochemical fields.
.alpha.-Galactosidase has the ability to sever the .alpha.-galactoside linkage in saccharides. If .alpha.-galactosidase of high purity can be obtained inexpensively, therefore, it will possibly find utility in the production of foodstuffs, medicines and drugs, reagents, etc. as well as in the production of beet sugar and in the determination of structures of chemical compounds.
The .alpha.-galactosidase which is produced by the culture of a microorganism occurs chiefly within the cells of the microorganism. To obtain .alpha.-galactosidase of high purity from the microorganism, therefore, the produced enzyme must be extracted from the cells and then subjected to a refining treatment.
Heretofore, it has been known to effect the extraction of .alpha.-galactosidase by crushing the cells containing the formed .alpha.-galactosidase by physical means. By this method, however, the recovery ratio of .alpha.-galactosidase is on such a low order as 40 to 50% and the purity itself is not sufficiently high. It has further been known to extract the .alpha.-galactosidase from the microorganic cells and refine it by chemical methods such as by precipitation of .alpha.-galactosidase using ammonium sulfate, tannin or organic solvent. The extracts obtained by these methods, however, contain impurities such as extraneous proteins, sugars, nucleic acids, fats, etc. still in high concentrations, making it necessary to incorporate in the operation an extra process for purification. None of these methods, therefore, has been able to recover and refine .alpha.-galactosidase efficiently in one process.
An object of this invention is to provide a method for easily recoveing .alpha.-galactosidase of high purity at a high yield from the .alpha.-galactosidase-containing liquid.